Stimulation of hair growth by peptide copper complexes

ABSTRACT

Peptide-copper complexes are disclosed which stimulate the growth of hair on warm-blooded animals. In one aspect of this invention, the peptide-copper complexes are dipeptides or tripeptides chelated to copper at a molar ratio ranging from about 1:1 to 3:1, with the second position of the peptide from the amino terminus being histidine, arginine or a derivative thereof. The peptide-copper complexes may be formulated for administration by, for example, topical application or injection. Any affliction associate with hair loss, including hair loss associated with both androgenetic and secondary alopecia, may be treated with the peptide-copper complexes of this invention.

This application is a continuation of application Ser. No. 08/638,889,filed Jul. 19, 1996, now abandoned, which is a continuation ofapplication Ser. No. 08/261,475, filed Jun. 17, 1994, now U.S. Pat. No.5,538,945.

TECHNICAL FIELD

This invention relates generally to peptide-copper complexes and, morespecifically, to compositions containing peptide-copper complexes forstimulating hair growth.

BACKGROUND OF THE INVENTION

Hair loss is a prevalent affliction of many humans, the most commonbeing androgenetic alopecia (AGA) where males lose scalp hair as theyget older (i.e., male pattern baldness). Other hair loss afflictionsinclude alopecia areata (AA), female pattern baldness and hair losssecondary to chemotherapy and/or radiation treatment (i.e., secondaryalopecia).

Hair is normally divided into two types: "terminal" and "vellus".Terminal hair is coarse, pigmented hair which arises from follicleswhich are developed deep within the dermis. Vellus hairs are typicallythin, non-pigmented hairs which grow from hair follicles which aresmaller and located superficially in the dermis. As alopecia progresses,there is a change from terminal to vellus type hair. Other changes thatcontribute to alopecia are alterations in the growth cycle of hair. Hairtypically progresses through three cycles, anagen (active hair growth),catagen (transition phase), and telogen (resting phase during which thehair shaft is shed prior to new growth). As baldness progresses, thereis a shift in the percentages of hair follicles in each phase with themajority shifting from anagen to telogen. The size of hair follicles isalso known to decrease while the total number remains relativelyconstant.

A variety of procedures and drugs have been utilized in an attempt totreat hair loss. A common technique involves hair transplantation.Briefly, plugs of skin containing hair are transplanted from areas ofthe scalp where hair was growing to bald or balding areas of the scalp.This procedure, however, is time-consuming and relatively painful. Otherapproaches include ultra-violet radiation and exercise therapy.

More recently, the stimulating hair growth has been achieved, althoughwith limited success, by drug therapy. One of the most well-recognizedhair-growth agents is sold under the tradename "Minoxidil", as disclosedin U.S. Pat. No. 4,596,812 assigned to Upjohn. However, while theresults generated through the use of Minoxidil have appeared promising,there is still a need in the art for improved compositions capable ofstimulating the growth of hair in warm-blooded animals. To this end,certain peptide-copper complexes have been found to be effectivehair-growth agents. For example, U.S. Pat. Nos. 5,177,061, 5,120,831 and5,214,032 disclose certain peptide-copper complexes which are effectivein stimulating the growth of hair in warm-blooded animals.

While significant progress has been made in the simulation ofhair-growth by drug treatment, there is still a need in the art forcompounds which have greater stimulatory effect on hair growth. Thepresent invention fulfills this need, while further providing otherrelated advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention is directed to peptide-coppercomplexes, and compositions containing the same, for stimulating thegrowth of hair in warm-blooded animals. Compositions of this inventioninclude one or more peptide-copper complexes in combination with anacceptable carrier or diluent. As used herein, the term "copper" is usedto designate copper(II) (i.e., Cu⁺²).

The peptide-copper complexes of this invention are administered to ananimal in need thereof in a manner which results in the application ofan effective amount of the peptide-copper complex. As used herein, theterm "effective amount" means an amount of the peptide-copper complexwhich stimulates hair growth associated with a hair-loss afflications(such as male pattern baldness) or caused by a hair-loss insult (such asradiation or chemotherapy). Thus, the peptide-copper complexes may beused propylactically, as well as therapeutically and cosmetically.Administration of the peptide-copper complexes is preferably by topicalapplication, although other avenues of administration may be employed,such as injection (e.g., intramuscular, intravenous, subcutaneous andintradermal). Typically, the peptide-copper complexes of this inventionare formulated as a solution, cream or gel for topical application, oras a solution for injection, and include one or more acceptable carriersor diluents.

As used herein, the term "peptide-copper complex" means a peptide havingat least two amino acids (or amino acid derivatives) chelated to copper,wherein the second amino acid from the amino terminus of the peptide ishistidine, arginine or a derivative thereof. Such peptide-coppercomplexes have the following general structure A:

A: [R₁ --R₂ ]:copper(II)

wherein:

R₁ is an amino acid or an amino acid derivative; and

R₂ is histidine, arginine or a derivative thereof.

The peptide-copper complexes of this invention have a ratio of peptideto copper ranging from about 1:1 to about 3:1, and more preferably fromabout 1:1 to about 2:1. In short, a component of the peptide occupies atleast one corrordination site of the copper ion, and multiple peptidesmay be chelated to a single copper ion.

In a preferred embodiment, the peptide-copper complex comprises afurther chemical moiety linked to the R₂ moiety of structure A by anamide or peptide bond. (i.e., --C(═O)NH--). In this embodiment, thepeptide-copper complex has the following structure B:

B: [R₁ --R₂ --R₃ ]:copper(II)

wherein:

R₁ is an amino acid or amino acid derivative;

R₂ is histidine, arginine or a derivative thereof; and

R₃ is a chemical moiety joined to R₂ by an amide bond.

In a further preferred embodiment, R₃ of structure B is at least oneamino acid joined to R₂ by a peptide bond. In this embodiment, thepeptide-copper complex has the following structure C:

C: [R₁ --R₂ --R₃ ]:copper(II)

wherein:

R₁ is an amino acid or amino acid derivative;

R₂ is histidine, arginine or a derivative thereof; and

R₃ is an amino acid or amino acid derivative joined to R₂ by a peptidebond, with the proviso that R₁ is not glycyl, alanyl, seryl or valylwhen R₂ is histidyl or (3-methyl)histidyl and R₃ is lysine,lysyl-prolyl-valyl-phenylalanyl-valine, lysyl-valyl-phenylalanyl-valine,lysyl-tryptophan, or lysyl-(glycyl)₁₋₂ -tryptophan, and with the furtherproviso that R₁ is not lysyl when R₂ is histidyl or (3-methyl)histidyland R₃ is glycine, glycyl-prolyl-valyl-phenylalanyl-valine,glycyl-valyl-phenylalanyl-valine, gylcyl-tryptophan, orglycyl-(glycyl)₁₋₂ -tryptophan.

In still a further embodiment of the present invention, an additionalchelating agent may be added to the peptide-copper complexes disclosedabove to form a ternary peptide-copper-chelating agent complex.

Other aspects of the present invention will become evident uponreference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to peptide-copper complexes which stimulatethe growth of hair on warm-blooded animals. Such complexes are typicallyadministered as a composition containing acceptable diluents and/orcarriers. Administration is preferably by topical application directlyto the area where stimulation of hair growth is desired, such as thescalp, although other routes of administration may be employed.

The peptide-copper complexes of this invention may be used to stimulatehair growth in animals (including humans) afflicted with androgeneticalopecia (AGA). Animals afflicted with this condition are usually male,and the condition results in the loss of scalp hair with age (alsocalled "male pattern baldness"). Thus, the peptide-copper complexes maybe administered in order to stimulate hair growth, thereby eliminatingor reducing the severity of hair loss and/or the speed at which AGAprogresses. Other hair loss afflictions include alopecia areata (AA),female pattern baldness and hair loss secondary to chemotherapy and/orradiation treatment (i.e., secondary alopecia). In the case of secondaryalopecia, the peptide-copper complexes may be used in advance of certainhair-loss insults, such as chemotherapy or radiation regiments, tostimulating hair growth prior to the insult and thereby reduce theamount of hair loss resulting therefrom.

As mentioned above, the peptide-copper complexes of the presentinvention have at least two amino acids (or amino acid derivatives), oneof which is histidine, arginine or a derivative thereof. In thiscontext, the peptide-copper complexes have structure A as identifiedabove. For example, when R₁ is an amino acid and R₂ is histidyl, or whenR₁ is an amino acid and R₂ is arginine, the peptide copper complex hasthe following structures D and E, respectively:

D: [(amino acid)-histidine]:copper(II)

E: [(amino acid)-arginine]:copper(II)

As used in structure A above, the terms "amino acid" and "amino acidderivative" are defined hereinbelow. An amino acid of this inventionincludes any carboxylic acid having an amino moiety, including (but notlimited to) the naturally occuring α-amino acids (in the followinglisting, the single letter amino acid designations are given inparentheses): alanine (A), arginine (R), asparagine (N), aspartic acid(D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G),histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M),phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan(W), tyrosine (Y) and valine (V). Other naturally occuring amino acidsinclude (but are not limited to) hydroxyproline and γ-carboxyglutumate.In a preferred embodiment, the amino acid is a naturally occuringα-amino acid having an amino moiety (i.e., the --NH₂ group, rather thana secondary amine, --NH--, such as present in proline) attached to theα-carbon of the amino acid which, when chelated to copper, occupies acoordination site thereof. As used herein, "hydrophillic amino acids"include (but are not limited to) the amino acids selected from K, R, H,D, E, N, Q, C, M, S and T.

An amino acid derivative of this invention includes any compound havingthe structure: ##STR1## wherein R is a derivative of a naturallyoccurring amino acid side chain. In one embodiment, R₁ and R₂ in theabove structure may be selected from hydrogen, a substituted orunsubstituted, straight chain, branched or cyclic, saturated orunsaturated alkyl moiety containing from 1-20 carbon atoms, and asubstituted or unsubstituted aryl moiety containing from 6-20 carbonatoms (including heteroaromatic moieties). In a preferred embodiment, R₁and R₂ may be selected from the chemical moieties identified in Table 1below.

                  TABLE 1                                                         ______________________________________                                        Amino Acid Derivatives                                                        ______________________________________                                         ##STR2##                                                                     ______________________________________                                    

Where R₂ =H or the following moieties:

--(CH₂)_(n) CH₃ where n=1-20

--(CH₂)_(n) CH(CH₃)(CH₂)_(m) CH₃ where n, m=0-20

(when n=0, m≠0 or 1 and when n=1, m≠0)

--(CH₂)_(n) NH₂ where n=1-20 (n≠4)

--(CH₂)_(n) CONH₂ where n=3-20

--(CH₂)_(n) COOH where n=3-20 ##STR3## where n=2-20 ##STR4## wheren=2-20 ##STR5## where n=2-20 --(CH₂)_(n) SH where n=2-20

--(CH₂)_(n) S(CH₂)_(m) CH₃ where n, m=1-20

(when n=2, m≠0)

--(CH₂)_(n) CH₂ OH where n=1-20

--(CH₂)_(n) CH(CH₃)OH where n=1-20

And where R₁ =H or the following moieties:

--(CH₂)_(n) CH₃ where n=0-20

--(CH₂)_(n) CH(CH₃)(CH₂)_(m) CH₃ where n, m=0-20

Histidine derivatives of this invention include compounds having thestructure: ##STR6## where n=1-20, and X and Y are independently selectedfrom alkyl moieties containing from 1-12 carbon atoms or an aryl moietycontaining from 6-12 carbon atoms. In preferred embodiments, n is 1, Xis methyl and Y is H (i.e., 3-methyl histidyl) or X is H and Y is methyl(i.e., 5-methyl histidine).

Similarly, arginine derivatives of this invention include compoundshaving the structure: ##STR7## where n=1-20 (excluding n=3).

In another embodiment of this invention, the peptide-copper complexes ofstructure A further comprise a chemical moiety linked to the R₂ moietyby an amide or peptide bond. (i.e., --C(═O)NH--). The peptide-coppercomplexes of this embodiment are depicted above as structure B. As usedherein, a chemical moiety (i.e., R₃) linked to the R₂ moiety by an amidebond includes any chemical moiety having an amino group capable offorming an amide linkage with the carboxyl terminus of R₂ (i.e., thecarboxyl terminus of histidine, arginine, or derivatives thereof).Suitable R₃ moieties include (but are not limited to) --NH₂, alkylaminomoieties having from 1-20 carbon atoms and arylamino moieties havingfrom 6-20 carbon atoms, as well as amino acids and derivatives thereof.As used herein, "alkylamino moieties" include alkyl moieties containingan amino moiety, wherein the alkyl moiety is as defined above, andincludes (but is not limited to) octyl amine and propyl amine.Similarly, "arylamino moieties" include aryl moieties containing anamino moiety, wherein the aryl moiety is as defined above, and includes(but is not limited to) benzylamine and benzyl-(CH₂)₁₋₁₄ -amine. Furtherexamples of suitable chemical moieties having amino groups capable offorming an amide linkage with the carboxyl terminus of R₂ includepolyamines such as spermine and sperimidine.

For example, in structure B when R₁ is an amino acid, R₂ is histidine orarginine, and R₃ is an amino moiety, the peptide-copper complex has thefollowing structures F and G, respectively:

F: [(amino acid)-histidine-NH₂ ]:copper(II)

G: [(amino acid)-arginine-NH₂ ]:copper(II)

Similarly, when R₁ is an amino acid, R₂ is histidine or arginine, and R₃is an alkylamino moiety, the peptide-copper complex has the followingstructures H and I, respectively:

H: [(amino acid)-histidine-NH-alkyl]:copper(II)

I: [(amino acid)-arginine-NH-alkyl]:copper(II)

In yet a further embodiment (as represented by structure C above), theR₃ moiety of structure B is at least one an amino acid or an amino acidderivative as defined above. In a preferred embodiment, R₃ is anaturally occuring α-amino acid joined to R₂ by a peptide bond. Forexample, when R₁ and R₃ of structure C are amino acids, and R₂ ishistidine or arginine, the peptide-copper complexes of this inventionhave the following structures J and K, respectively:

J: [(amino acid)-histidine-(amino acid)]:copper(II)

K: [(amino acid)-arginine-(amino acid)]:copper(II)

It should be understood that while only a single amino acid is depictedin the R₃ position of structures H and I, other chemical moieties mayalso be present, including additional amino acids and/or amino acidderivatives. For example, R₃ in structures H and I may be a peptide suchas phenylalanine-phenylalanine, (glycyl)_(n) -tryptophan where n=1-4,prolyl-X₁ -phenylalanyl-X₂ or X₁ -phenylalanyl-X₂ where X₁ and X₂ areselected from valine, alanine and glycine.

The peptides of the peptide-copper complexes of this invention maygenerally be classified as dipeptides (i.e, structure A), dipeptideswith a chemical moiety attached to the carboxyl terminus via an amidebond (i.e., structure B) or as tripeptides (i.e., structure C above). Inthe case of peptide-copper complexes of structures B and C, additionalchemical moieties, including amino acids, may be joined to the dipeptideor tripeptide to yield peptides containing four or more amino acids. Forpurpose of illustratation, Table 2 presents various representativeexamples of peptide-copper complexes of this invention.

                  TABLE 2                                                         ______________________________________                                        Representative Peptide-Copper Complexes                                       ______________________________________                                        Structure A:                                                                  glycyl-histidine:copper                                                                       alanyl-histidine:copper                                       glycyl-(3-methyl)histidine:                                                                   alanyl-(3-methyl)histidine:copper                             copper                                                                        glycyl-(5-methyl)histidine:                                                                   alanyl-(5-methyl)histidine:copper                             copper                                                                        glycyl-arginine:copper                                                                        alanyl-arginine:copper                                        (N-methyl)glycine-histidine:                                                                  (N-methyl)glycine-arginine:copper                             copper                                                                        Structure B:                                                                  glycyl-histidyl-NH.sub.2 :copper                                                              glycyl-arginyl-NH.sub.2 :copper                               glycyl-(3-methyl)histidyl-NH.sub.2 :                                                          alanyl-3-methyl)histidyl-NH.sub.2 :copper                     copper                                                                        glycyl-arginyl-NH.sub.2 :copper                                                               alanyl-arginyl-NH.sub.2 :copper                               (N-methyl)glycine-histidyl-                                                                   (N-methyl)glycine-arginyl-NH.sub.2 :copper                    NH.sub.2 :copper                                                              glycyl-histidyl-NHoctyl:copper                                                                glycyl-arginyl-NHoctyl:copper                                 Structure C:                                                                  glycyl-histidyl-lysine:copper                                                                 glycyl-arginyl-lysine:copper                                  glycyl-(3-methyl)histidyl-lysine:                                                             glycyl-(5-methyl)histidyl-lysine:copper                       copper                                                                        alanyl-histidyl-lysine:copper                                                                 alanyl-arginyl-lysine:copper                                  alanyl-(3-methyl)histidyl-lysine:                                                             alanyl-(5-methyl)histidyl-lysine:copper                       copper                                                                        glycyl-histidyl-phenylalanine:                                                                glycyl-arginyl-phenylalanine:copper                           copper                                                                        glycyl-(3-methyl)histidyl-                                                                    glycyl-(5-methyl)histidyl-                                    phenylalanine:copper                                                                          phenylalanine:copper                                          alanyl-histidyl-phenylalanine:                                                                alanyl-arginyl-phenylalanine:copper                           copper                                                                        alanyl-(3-methyl)histidyl-                                                                    alanyl-(5-methyl)histidyl-                                    phenylalanine:copper                                                                          phenylalanine:copper                                          glycyl-histidyl-lysyl-                                                                        glycyl-arginyl-lysyl-phenylalanyl-                            phenylalanyl-phenylalanyl:                                                                    phenylalanyl:copper                                           copper                                                                        glycyl-(3-methyl)histidyl-lysyl-                                                              glycyl-(5-methyl)histidyl-lysyl-                              phenylalanyl-phenylalanyl:                                                                    phenylalanyl-phenylalanyl:copper                              copper                                                                        (N-methyl)glycyl-histidyl-lysine:                                                             (N-methyl)glycyl-arginyl-lysine:copper                        copper                                                                        ______________________________________                                    

Further examples of peptide-copper complexes of this invention aredisclosed in U.S. Pat. Nos. 5,118,665 and 5,164,367, as well as U.S.Pat. Nos. 4,760,051; 4,665,054; 4,877,770; 5,177,061; 4,810,693;4,767,753; 5,135,913; 5,023,237; 5,059,588 and 5,120,831, all of whichare incorporated herein by reference in their entirety. Thus, thepeptide-copper complexes disclosed in the above U.S. patents may be usedto stimulate hair growth in animals (including humans) afflicted withandrogenetic alopecia (AGA) or male pattern baldness, therebyeliminating or reducing the severity of hair loss and/or the speed atwhich AGA progresses. These peptide-copper complexes may also by used totreat other hair loss afflictions, include alopecia areata, femalepattern baldness and hair loss secondary to chemotherapy and/orradiation treatment (i.e., secondary alopecia). In the case of secondaryalopecia, the peptide-copper complexes may be used to stimulate hairgrowth prior to a insults which normally result in hair loss, such aschemotherapy or radiation regiments. Thus, the peptide-copper complexesof this invention may be used to prevent hair loss.

In the practice of this invention, the molar ratio of peptide to copperis greater than zero to one (e.g., 0.1:1, 0.2:1, etc.). The molar ratioof peptide to copper will depend, in part, on the number of coppercoordination sites that are occupied by the peptide. In a preferredembodiment, the molar ratio of peptide to copper ranges from about 1:1to 3:1, and more preferably from about 1:1 to 2:1. For example, in thecase of a tripeptide (such as GHF:copper), the preferred ratio ofpeptide to copper ranges from 1:1 to 2:1, with each tripeptide occupyingthree coordination sites of the copper. Similarly, with a dipeptide(such as GH:copper), the preferred ratio of peptide to copper rangesfrom 1:1 to 3:1, with each dipeptide occupying two coordination sites ofcopper ion.

In another embodiment of this invention, a chelating agent may be addedto the peptide-copper complex to form a ternary peptide-copper-chelatingagent complex. Suitable chelating agents include imidazole orimidazole-containing compounds, such as histidine, and sulfur containingamino acids, such as cysteine or methionine. Thus, if the peptide-coppercomplex is GHF:copper, histidine may be added to yield the ternarycomplex GHF:copper:histidine. However, to form such a ternary complex,the molar ratio of copper to peptide to chelating agent must beconsidered. For example, if the ratio of peptide to copper is 2:1, theaddition of a chelating agent to the peptide-copper complex, althoughpossible, is difficult due to site occupancy by the peptide. However, bymaintaining the ratio of peptide to copper near 1:1, a chelating groupmay readily be added to form the ternary complex. Thus, the preferredpeptide to copper to chelating agent ratio is about 1:1:1.

While the chiral amino acids of the present invention (particularly theamino acids) have not been specifically designated, the presentinvention encompasses both the naturally occuring L-form, as well as theD-form. For example, any of the naturally occuring L-amino acids (oramino acid derivatives) disclosed herein may be replaced by acorresponding D-amino acid (or amino acid derivative).

In the practice of this invention, it is critical that the secondposition of the peptide (i.e., R₂ of structures A, B and C) is eitherhistidine, arginine or a derivative thereof. It is believed that thesuperior effect of the peptide-copper complexes of the present inventionis achieved, at least in part, by the binding of copper by an aminomoiety of the amino acid side chain of histidine, arginine or derivitivethereof. For example, in the case of histidine, an amine group of thehistidine imidazole ring occupies a coordination site of the copper(i.e., the residual valencies or unshared electrons of the amine groupare shared with copper). In the case of arginine, an amine group of theamino acid side chain similarly occupies a coordination site of copper.The binding of R₂ to the copper atom is preferably combined with thecoordination of an amine group from the R₁ moiety of structures A, B andC, to yield the peptide-copper complex. Thus, a peptide of thisinvention chelates copper by donating the R₂ amine group, and preferablyboth the R₁ and R₂ amine groups, to the peptide-copper complex. Thepeptide-copper complexes of structures B and C can further occupyadditional coordination sites on copper. Specifically, the amine groupof the amide bond of structure B and the peptide bond of structure C canoccupy yet a further coordination sites.

As mentioned above, the peptide-copper complexes of this invention haveutility as hair growth agents. More particularly, the peptide-coppercomplexes stimulates hair growth on warm-blooded animals. Thus, thepeptide-copper complexes may be used to treat a variety of diseasesstates associated with hair loss, including (but not limited to)androgenetic alopecia (also know as male pattern baldness), alopeciaareata and female pattern baldness. In these instances, thepeptide-copper complexes stimulates the growth of hair after the onsetof the hair-loss affliction. Alternatively, the peptide-copper complexesmay be administered prophylactically for conditions such as secondaryalopecia. For example, the complexes may be administered prior to aninsult which normally results in hair loss, such as chemotherapy and/orradiation treatment. Thus, the peptide-copper complexes of thisinvention can be used to prevent hair loss.

Administration of the peptide-copper complexes of the present inventionmay be accomplished in any manner which will result in the delivery ofan effective amount or dose of the peptide-copper complex to the animal,including delivery to the hair follicles. For example, administrationmay be by topical application directly to the scalp, or other area wherehair stimulation is desired (hereinafter "the treatment area").Alternatively, administration may also be accomplished by injection(such as intradermal injection) into the treatment area, including thescalp. Typically, the peptide-copper complexes are formulated as acomposition containing the peptide-copper complex in combination with onor more acceptable carriers or diluents, including formulations whichprovide for the sustained release of the peptide-copper complexes overtime.

In one embodiment, the peptide-copper complexes are formulated forintradermal injection to the treatment area. In such instances, suchformulations preferably contain one or more peptide-copper complexes ofthis invention in combination with a suitable vehicle for intradermalinjection, with the peptide-copper complex present in the composition ata concentration ranging from 100 μg to 2000 μg per 0.1 ml vehicle (i.e.,1.0 mg/ml to 20 mg/ml). Suitable vehicles for intradermal injectioninclude (but are not limited to) saline and sterile water.

In another embodiment, the peptide-copper complexes are formulated fortopical administration. Suitable topical formulations include one ormore peptide-copper complexes in the form of a liquid, lotion, cream orand gel. Topical administration may be accomplished by applicationdirectly on the treatment area. For example, such application may beaccomplished by rubbing the formulation (such as a lotion or gel) ontothe skin of the treatment area, or by spray application of a liquidformulation onto the treatment area. Any quantity of the topicalformulation sufficient to accelerate the rate of hair growth or preventsubsequent hair loss is effective, and treatment may be repeated asoften as the progress of hair growth indicates. Preferable, the topicalcompositions of this invention contain one or more peptide-coppercomplexes in an amount ranging from 0.1% to 20% by weight of thecomposition, and more preferably from 0.1% to 5% by weight of thecomposition.

In addition to carriers and diluents, the peptide-copper complexes mayalso be formulated to contain additional ingredients such as penetrationenhancement agents and/or surface active agents. For example, topicalformulations may contain 0.5% to 10% of one or more surface activeagents (also called emulsifying agents). Non-ionic surface active agentsand ionic surface active agents may be used for the purposes of thepresent invention. Examples of suitable non-ionic surface active agentsare nonylphenoxypolyethoxy ethanol (Nonoxynol-9), polyoxyethylene oleylether (Brij-97), various polyoxyethylene ethers (Tritons), and blockcopolymers of ethylene oxide and propylene oxide of various molecularweights (such as Pluronic 68). Examples of suitable ionic surface activeagents include sodium lauryl sulfate and similar compounds. Penetrationenhancing agents may be also be present in topical formulations.Suitable penetration enhancing agents include dimethyl sulfoxide (DMSO),urea and substituted urea compounds. In the case of a liquid formulationfor topical administration, the concentration of the penetratingenhancing agent (such as DMSO) may range from 30% to 80% of liquidformulation.

The balance of the topical formulations may include inert,physiologically acceptable carriers or diluents. Suitable carriers ordiluents include, but are not limited to, water, physiological saline,bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol),petrolatum based creams (e.g., USP hydrophilic ointments and similarcreams, Unibase, Parke-Davis, for example), various types ofpharmaceutically acceptable gels, and short chain alcohols and glycols(e.g., ethyl alcohol and propylene glycol). In another embodiment of theinvention, topical formulations may also contain the peptide-coppercomplex encapsulated in liposomes to aid in the delivery of thepeptide-copper complex to the hair follicle. Alternatively, thepeptide-copper complex may be formulated in an instrument to deliver thecompound via iontophoresis.

The peptide-copper complexes of this invention exhibit superior skinpermeability when applied topically. This results in a greater effectivedose to the treatment area, and thus correspondingly greater simulationof hair growth. In the practice of this invention, hydrophobic aminoacids or amino acid derivatives are preferably used for administrationby injection (such as intradermal injection), while hydrophilic aminoacids or amino acid derivatives are used for topical administration.While the use of hydrophobic amino acids or amino acid derivativesgenerally enhance activity of the copper-peptide complexes of thisinvention, the use of hydrophilic amino acids or amino acids derivativesfor topical administration is prefered due to the enhanced skinpermeability associated therewith.

For purpose of illustration, Table 3 presents examples of suitabletopical formulations within the context of the present invention. Asused below, "% (w/w)" represents the weight percentage of a componentbased on the total weight of the formulation:

                  TABLE 3                                                         ______________________________________                                        Representative Topical Formulatoins                                           ______________________________________                                        Preparation A:                                                                Peptide-Copper Complex 1.0%    (w/w)                                          Hydroxy Ethyl Cellulose                                                                              3.0%    (w/w)                                          Propylene Glycol       20.0%   (w/w)                                          Nonoxynol-9            3.0%    (w/w)                                          Benzyl Alcohol         2.0%    (w/w)                                          Aqueous Phosphate Buffer (0.2N)                                                                      71.0%   (w/w)                                          Preparation B:                                                                Peptide-Copper Complex 1.0%    (w/w)                                          Nonoxynol-9            3.0%    (w/w)                                          Ethyl Alcohol          96.0%   (w/w)                                          Preparation C:                                                                Peptide-Copper Complex 5.0%    (w/w)                                          Ethyl Alcohol          47.5%   (w/w)                                          Isopropyl Alcohol      4.0%    (w/w)                                          Propylene Glycol       20.0%   (w/w)                                          Lanoeth-A              1.0%    (w/w)                                          Water                  27.5%   (w/w)                                          Preparation D:                                                                Peptide-Copper Complex 5.0%    (w/w)                                          Sterile Water          95.0%   (w/w)                                          Preparation E:                                                                Peptide-Copper Complex 2.5%    (w/w)                                          Hydroxypropyl Cellulose                                                                              2.0%    (w/w)                                          Glycerine              20.0%   (w/w)                                          Nonoxynol-9            3.0%    (w/w)                                          Sterile Water          72.5%   (w/w)                                          Preparation F                                                                 Peptide-Copper Complex 0.5%    (w/w)                                          Sterile Water          16.5%   (w/w)                                          Propylene Glycol       50.0%   (w/w)                                          Ethanol                30.0%   (w/w)                                          Nonoxynol-9            3.0%    (w/w)                                          Preparation G:                                                                Peptide-Copper Complex 5.0%    (w/w)                                          Sterile Water          10.0%   (w/w)                                          Hydroxypropyl Cellulose                                                                              2.0%    (w/w)                                          Propylene Glycol       30.0%   (w/w)                                          Ethanol                50.0%   (w/w)                                          Nonoxynol-9            3.0%    (w/w)                                          ______________________________________                                    

The peptides of the present invention may be synthesized by eithersolution or solid phase techniques known to one skilled in the art ofpeptide synthesis. The general procedure involves the stepwise additionof protected amino acids to build up the desired peptide sequence. Theresulting peptide may then be complexed to copper (at the desired molarratio of peptide to copper) by dissolving the peptide in water, followedby the addition of copper chloride and adjusting the pH. A more detaileddisclosure directed to the synthesis of the peptide-copper complexes ofthis invention, as well as the activity certain representativepeptide-copper complexes, are presented below.

EXAMPLES

The following examples are offered by way of illustration, and not byway of limitation. To summarize the examples that follow, Example 1discloses the general preparation of peptide-copper complexes of thepresent invention by chelating a peptide to copper in an aqueoussolution. Examples 2-10 disclose the synthesis of peptides which may bechelated to copper to yield peptide-copper complexes. Examples 11-16disclose the ability of representative peptide-copper complexes of thisinvention to stimulate hair growth.

Source of Chemicals

Chemicals and peptide intermediates utilized in the following examplesmay be purchased from a number of suppliers, including: Sigma ChemicalSo., St. Louis, Mo.; Peninsula Laboratories, San Carlos, Calif.; AldrichChemical Company, Milwaukee, Wis.; Vega Biochemicals, Tucson, Ariz.;Pierce Chemical Co., Rockford, Ill.; Research Biochemicals, Cleveland,Ohio; Van Waters and Rogers, South San Francisco, Calif.; and Bachem,Inc., Torrance, Calif.

EXAMPLE 1 Preparation of Peptide-Copper Complex

The peptide-copper complexes of the present invention may be synthesizedby dissolving the peptide in distilled water, followed by the additionof copper chloride (e.g., 99.999% available from Chemical Dynamics,N.J.) and then adjusting the pH of the solution to about 7.0. Forexample, copper complexes of glycyl-L-histidyl-L-phenylalanine (GHF)with a molar ratio of peptide to copper of 1:1, 2:1, or greater (e.g.,3:1), may be prepared by dissolving a given weight of GHF in distilledwater (e.g., 50 mg/ml), and adding the desired molar amount of purifiedcopper-chloride. The pH of the resulting peptide solution is thenadjusted to about 7.0 by the addition of, for example, a sodiumhydroxide solution. Alternatively, copper salts other than copperchloride may be used, for example, copper acetate, copper sulfate orcopper nitrate.

EXAMPLE 2 Synthesis of Glycyl-L-Histidyl-L-Caprolactam

L(-)-3-amino-ε-caprolactam was dissolved in tetrahydrofuran (THF) thencoupled with N^(a) -t-butyloxycarbonyl-N^(im)-benzyloxycarbonyl-L-histidine (N^(a) -BOC-N^(im) -CBZ-L-histidine)using isobutyl chloroformate and N-methylmorpholine in THF. After twohours at -20° C. and an additional hour at ambient temperature, thereaction was quenched with 2N aqueous potassium bicarbonate. Thisproduct was extracted into ethyl acetate, washed with 1M aqueous citricacid, and saturated sodium bicarbonate. The organic phase was dried overanhydrous sodium sulfate. Filtration and evaporation gave N.sup.α-BOC-N^(im) -CBZ-L-histidyl-L-caprolactam.

The above compound was dissolved in 30% trifluoroacetic acid indichloromethane for 30 minutes, then evaporated, forming N^(im)-benzyloxycarbonyl-L-histidyl-L-caprolactam. This was then dissolved intetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine andbenzyloxycarbonyl-glycine were added to formbenzyloxycarbonyl-glycyl-N^(im)-benzyloxycarbonyl-L-histidyl-L-caprolactam. This product wasrecrystallized once from ethyl acetate then dissolved in acetic acid andhydrogenated overnight in the presence of 10% Pd-C catalyst. Theresultant glycyl-L-histidyl-L-caprolactam was lyophilized from waterseveral times, then purified by liquid chromatography on a C-18reverse-phase column to yield the peptide as a diacetate salt.

EXAMPLE 3 Synthesis of L-Alanyl-L-Histidyl-L-Phenylalanine

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (9.74 g,25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.3 mmol). After 2 min. phenylalanine benzyl ester tosylate(10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15°C. for 1.5 h and then allowed to warm to 0° C. At this time the reactionwas quenched by the addition of 2M aqueous potassium bicarbonate. Theproducts were extracted with ethyl acetate (3×150 mL). The combinedextracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO₃(3×100 mL), water, and brine. The resulting solution was dried oversodium sulfate, filtered, and evaporated to give 13.7 g (87%) of theblocked dipeptide as a white semi-solid (R_(f) =0.75, 10%methanol/dichloromethane), which was used in the followingtransformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (12.9 g, 20.6mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×150 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 13.3 g (ca.100%+entrained solvent) of the free-amino compound as a white solid:R_(f) =0.49 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-L-alanine (6.03 g, 27.0 mmol) andN-methylmorpholine (3.3 mL, 3.0 g, 29.7 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (3.7 mL, 3.9 g, 28.4 mmol).After 2 min. a solution of the suitably protected dipeptide (11.4 g,21.8 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixturewas stirred at -15° C. for 1.5 h and then allowed to warm to 0° C. Atthis time the reaction was quenched by the addition of 2M aqueouspotassium bicarbonate. The products were extracted with ethyl acetate(3×100 mL). The combined extracts were washed with 1M citric acid (3×100mL), water, 2M KHCO₃ (3×100 mL), water, and brine. The resultingsolution was dried over sodium sulfate, filtered, and evaporated to givethe blocked tripeptide as a white solid (R_(f) =0.55, 10%methanol/dichloromethane), which was recrystallized from 95% ethanol togive 12.6 g (79%) of a free-flowing white powder: mp 147-147.5° C.;Anal. Calcd. for C₄₁ H₄₀ N₅ O₈ : C, 67.39; H, 5.52; N, 9.58. Found: C,66.78; H, 5.64; N, 9.24.

To a suspension of the blocked tripeptide (12.6 g, 17.6 mmol) in ethanol(150 mL) was added water, until the mixture became very turbid (about150 mL). The resulting mixture was shaken with palladium chloride (1.56g, 8.8 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. Thecatalyst was removed by filtration through a plug of Celite® and thefiltrate was concentrated to remove volatile organic materials. Theremainder was lyophilized to give 8.30 g of white powder. This materialwas dissolved in water, filtered through a 0.2 m nylon membrane andlyophilized to give 6.27 g (87%) of the desired tripeptidedihydrochloride as a free-flowing white powder: [a]_(D) 5.1° (c 2.0,water); ¹ H NMR (500 MHz, DMSO-d₆) d 8.71 (1H, d, J=7.9), 8.49 (1H, d,J=7.8), 8.21 (1H, s), 7.30-7.22 (4H, m), 7.20-7.15 (1H, m), 7.12 (1H,s), 4.54 (1H, br q, J=7.1), 4.37 (1H, m), 3.86 (1H, q, J=6.8), 3.12 (1H,dd, J=4.3, 13.8), 3.05-2.90 (2H, m), 2.88 (1H, dd, J=9.5, 13.8), 1.27(3H, d, J=6.8); ¹³ C NMR (125 MHz, DMSO-d₆) d 173.5, 169.9, 169.5,138.1, 134.2, 130.5, 129.2, 128.2, 126.4, 117.8, 54.4, 52.5, 48.0, 36.8,28.5, 17.2.

EXAMPLE 4 Synthesis of Glycyl-L-Histidyl-L-Glutamic Acid

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (9.74 g,25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.3 mmol). After 2 min. glutamic acid dibenzyl estertosylate (12.5 g, 25.0 mmol) was added. The reaction mixture was stirredat -15° C. for 1.5 h and then allowed to warm to 0° C. At this time thereaction was quenched by the addition of 2M aqueous potassiumbicarbonate. The products were extracted with ethyl acetate (3×150 mL).The combined extracts were washed with 1M citric acid (3×100 mL), water,2M KHCO₃ (3×100 mL), water, and brine. The resulting solution was driedover sodium sulfate, filtered, and evaporated to give 15.2 g (87%) ofthe blocked dipeptide as a white semi-solid (R_(f) =0.74, 10%methanol/dichloromethane), which was used in the followingtransformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (15.1 g, 21.6mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×150 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 14.8 g (ca.100%+entrained solvent) of the free-amino compound as a white solid:R_(f) =0.48 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (5.23 g, 25.0 mmol) andN-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol).After 2 min. a solution of the suitably protected dipeptide (12.9 g,21.6 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixturewas stirred at -15° C. for 1.5 h and then allowed to warm to 0° C. Atthis time the reaction was quenched by the addition of 2M aqueouspotassium bicarbonate. The products were extracted with ethyl acetate(3×100 mL). The combined extracts were washed with 1M citric acid (3×100mL), water, 2M KHCO₃ (3×100 mL), water, and brine. The resultingsolution was dried over sodium sulfate, filtered, and concentrated to asyrup, which was diluted with absolute ethanol, and kept overnight at-20° C. The resulting precipitate was collected on a filter to afford9.93 g (58%) of the blocked tripeptide as a white solid (R_(f) =0.58,10% methanol/dichloromethane): mp 114-116° C. Anal. Calcd. for C₄₃ H₄₃N₅ O₁₀ : C, 65.39; H, 5.49; N, 8.87. Found: C, 64.93; H, 5.56; N, 8.41.

To a suspension of the blocked tripeptide (9.6 g, 12.2 mmol) in ethanol(150 mL) was added water, until the mixture became very turbid (about150 mL). The resulting mixture was shaken with palladium chloride (2.22g, 12.5 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. Thecatalyst was removed by filtration through a plug of Celite® and thefiltrate was concentrated to remove volatile organic materials. Theremainder was lyophilized to give 4.72 g of white powder. This materialwas dissolved in water, filtered through a 0.2 m nylon membrane andlyophilized to give 4.64 g (93%) of the desired tripeptidedihydrochloride as a free-flowing white powder: [a]_(D) -16.6° (c 2.0,water); ¹ H NMR (500 MHz, D₂ O) d 8.65 (1H, s), 7.35 (1H, s), 4.77 (1H,m), 4.46 (1H, m), 3.88 (2H, s), 3.28 (1H, dd, J=15.3, 6.1), 3.21 (1H,dd, J=15.3, 8.0), 2.47 (2H, m), 2.21 (2H, m), 2.00 (2H, m); ¹³ C NMR(125 MHz, D₂ O) d 179.9, 177.3, 174.3, 169.8, 136.5, 130.8, 120.4, 55.6,54.9, 43.3, 32.8, 29.3, 28.5; Anal. Calcd for C₁₃ H₂₁ Cl₂ N₅ O₆ : C,37.69; H, 5.11; N, 16.91; Cl, 17.12. Found: C, 37.23; H, 5.07; N, 16.01;Cl, 17.95.

EXAMPLE 5 Synthesis of Glycyl-L-Histidyl-L-Phenylalanine

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (9.74 g,25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.3 mmol). After 2 min. phenylalanine benzyl ester tosylate(10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15°C. for 1.5 h and then allowed to warm to 0° C. At this time the reactionwas quenched by the addition of 2M aqueous potassium bicarbonate. Theproducts were extracted with ethyl acetate (3×150 mL). The combinedextracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO₃(3×100 mL), water, and brine. The resulting solution was dried oversodium sulfate, filtered, and evaporated to give 13.0 g (83%) of theblocked dipeptide as a white semi-solid (R_(f) =0.79, 10%methanol/dichloromethane), which was used in the followingtransformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (12.9 g, 20.6mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×150 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 12.3 g (ca.100%+entrained solvent) of the free-amino compound as a white solid:R_(f) =0.50 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (5.23 g, 25.0 mmol) andN-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol).After 2 min. a solution of the suitably protected dipeptide (10.8 g,20.6 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixturewas stirred at -15° C. for 1.5 h and then allowed to warm to 0° C. Atthis time the reaction was quenched by the addition of 2M aqueouspotassium bicarbonate. The products were extracted with ethyl acetate(3×100 mL). The combined extracts were washed with 1M citric acid (3×100mL), water, 2M KHCO₃ (3×100 mL), water, and brine. The resultingsolution was dried over sodium sulfate, filtered, and evaporated to give14.0 g (95%) of the blocked tripeptide as a white solid (R_(f) =0.64,10% methanol/dichloromethane), which was recrystallized from absoluteethanol to give a free-flowing white powder.

To a suspension of the blocked tripeptide (6.0 g, 8.3 mmol) in ethanol(150 mL) was added water, until the mixture became very turbid (about150 mL). The resulting mixture was shaken with palladium chloride (1.47g, 8.3 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. Thecatalyst was removed by filtration through a plug of Celite® and thefiltrate was concentrated to remove volatile organic materials. Theremainder was lyophilized to give 1.46 g of white powder. This materialwas dissolved in water, filtered through a 0.2 m nylon membrane andlyophilized to give 1.38 g (38%) of the desired tripeptidedihydrochloride as a free-flowing white powder: [a]_(D) -7.5° (c 1.0,water); ¹ H NMR (500 MHz, D₂ O) d 8.59 (1H, s), 7.39-7.25 (5H, m), 7.21(1H, s), 4.70 (1H, br t, J=7), 3.80 (2H, s), 3.24 (1H, dd, J=14.0, 5.5),3.16 (1H, dd, J=15.4, 6.9), 3.10 (1H, dd, J=15.4, 7.4), 3.03 (1H, dd,J=14.0, 9.1); ¹³ C NMR (125 MHz, DMSO-d₆) d 172.7, 169.5, 166.0, 137.6,133.3, 129.2, 128.9, 128.3, 126.5, 116.8, 53.9, 51.8, 40.1, 36.4, 27.3.

EXAMPLE 6 Synthesis of Glycyl-L-Histidyl-L-Lysyl-Phenylalanine

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-lysine (9.5 g, 25.0mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.7 mmol). After 2 min. phenylalanine benzyl ester tosylate(10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15°C. for 1.5 h and then allowed to warm to 0° C. At this time the reactionwas quenched by the addition of 2M aqueous potassium bicarbonate. Theproducts were extracted with ethyl acetate (3×150 mL). The combinedextracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO₃(3×100 mL), water, and brine. The resulting solution was dried oversodium sulfate, filtered, and evaporated to give 17.76 g (ca.100%+entrained solvent) of the blocked dipeptide as a white solid (R_(f)=0.84, 10% methanol/dichloromethane), which was used in the followingtransformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (15.4 g, 25.0mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×100 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 15.8 g (ca.100%+entrained solvent) of the free-amino compound as a whitesemi-solid: R_(f) =0.55 (10% methanol/dichloromethane).

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (9.74 g,25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.7 mmol). After 2 min. a solution of the suitably protecteddipeptide (12.9 g, 25.0 mmol) in tetrahydrofuran (30 mL) was added. Thereaction mixture was stirred at -15° C. for 1.5 h and then allowed towarm to 0° C. At this time the reaction was quenched by the addition of2M aqueous potassium bicarbonate. The products were extracted with ethylacetate (3×150 mL). The combined extracts were washed with 1M citricacid (3×100 mL), water, 2M KHCO₃ (3×100 mL), water, and brine. Theresulting solution was dried over sodium sulfate, filtered, andevaporated to give 20.58 g (93%) of the blocked tripeptide as a whitesemi-solid (R_(f) =0.67, 10% methanol/dichloromethane), which was usedin the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected tripeptide (20.5 g, 23.1mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×150 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 20.5 g (ca.100%+entrained solvent) of the free-amino compound as a white solid:R_(f) =0.51 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (7.24 g, 34.6 mmol) andN-methylmorpholine (4.2 mL, 3.9 g, 38.1 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (4.7 mL, 5.0 g, 36.3 mmol).After 2 min. a solution of the suitably protected tripeptide (18.2 g,23.1 mmol) in 1:1 tetrahydrofuran/dimethylformamide (50 mL) was added.The reaction mixture was stirred at -15° C. for 1.5 h and then allowedto warm to 0° C. At this time the reaction was quenched by the additionof 2M aqueous potassium bicarbonate. The products were extracted withethyl acetate (3×150 mL). The combined extracts were washed with 1Mcitric acid (3×100 mL), water, 2M KHCO₃ (3×100 mL), water, and brine.The resulting solution was dried over sodium sulfate, filtered, andevaporated to give 21.6 g (95%) of the blocked tetrapeptide as a whitesolid (R_(f) =0.85, 10% methanol/dichloromethane), which was used in thefollowing transformation without further purification.

To a suspension of the blocked tetrapeptide (21.5 g, 21.9 mmol) inethanol (150 mL) was added water, until the mixture became very turbid(about 125 mL). The resulting mixture was shaken with palladium chloride(3.89 g, 21.9 mmol) under an atmosphere of hydrogen (5 atm) for 16 h.The reaction mixture became clear within about 1/2 h, which may indicatecompletion of the reaction. The catalyst was removed by filtration andthe filtrate was evaporated to give 13.7 g of colorless semi-solid. Thismaterial was dissolved in water and lyophilized to give 11.5 g (94%) ofthe desired tetrapeptide dihydrochloride as a free-flowing white powder:[a]_(D) -12.4° (c 2.0, H₂ O); ¹ H NMR (500 MHz, D₂ O ) d 8.72 (1H, d,J=7.7), 8.40 (1H, d, J=7.8), 8.00 (1H, s), 7.30-7.19 (5H, m), 7.01 (1H,s), 4.62 (1H, br q, J=4.7), 4.44 (1H, m), 4.22 (1H, br q, J=4.9), 3.58(2H, s), 3.10-2.90 (4H, m), 2.72 (2H, t, J=7.3), 1.65-1.20 (6H, m).

EXAMPLE 7 Synthesis ofGlycyl-L-Histidyl-L-Lysyl-L-Phenylalanyl-L-Phenylalanine

To a stirred solution of N^(a) -BOC-L-phenylalanine (10.6 g, 40.0 mmol)and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) in tetrahydrofuran (50mL) at -15° C. was added isobutyl chloroformate (5.5 mL, 5.7 g, 42.0mmol). After 2 min. a solution prepared by mixing phenylalanine benzylester tosylate (17.1 g, 40.0 mmol), tetrahydrofuran (50 mL), andN-methylmorpholine (4.4 mL, 4.0 g, 40.0 mmol) was added. The reactionmixture was stirred at -15° C. for 1.5 h and then allowed to warm to 0°C. At this time the reaction was quenched by the addition of 2M aqueouspotassium bicarbonate. The products were extracted with ethyl acetate(3×150 mL). The combined extracts were washed with 1M citric acid (3×100mL), water, 2M KHCO₃ (3×100 mL), water, and brine. The resultingsolution was dried over sodium sulfate, filtered, and evaporated to give19.8 g (98%) of the blocked dipeptide as a white solid (R_(f) =0.98, 10%methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected dipeptide (19.7 g, 39.2mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×100 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 19.3 g (ca.100%+entrained solvent) of the free-amino compound: R_(f) =0.65 (10%methanol/dichloromethane).

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-lysine (15.2 g, 40.0mmol) and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) intetrahydrofuran (100 mL) at -15° C. was added isobutyl chloroformate(5.5 mL, 5.7 g, 42.0 mmol). After 2 min. the protected dipeptide (15.8g, 39.2 mmol) was added. The reaction mixture was stirred at -15° C. for1.5 h and then allowed to warm to 0° C. At this time the reaction wasquenched by the addition of 2M aqueous potassium bicarbonate. Theproducts were extracted with ethyl acetate (3×150 mL). The combinedextracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO₃(3×100 mL), water, and brine. The resulting solution was dried oversodium sulfate, filtered, and evaporated to give 29.9 g (98%) of theblocked tripeptide as a white solid (R_(f) =0.84, 10%methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tripeptide (15.4 g, 25.0mmol) in 35% trifluoroacetic acid/dichloromethane (300 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×100 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 28.7 g (ca.100%+entrained solvent) of the free-amino compound as a fluffy whitesolid: R_(f) =0.72 (10% methanol/dichloromethane).

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (15.6 g,40.0 mmol) and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) intetrahydrofuran (80 mL) at -15° C. was added isobutyl chloroformate (5.5mL, 5.7 g, 42.0 mmol). After 2 min. a solution of the suitably protectedtripeptide (12.9 g, 25.0 mmol) in dimethylformamide (50 mL) was added.The reaction mixture was stirred at -15° C. for 1.5 h and then allowedto warm to 0° C. At this time the reaction was quenched by the additionof 2M aqueous potassium bicarbonate. The products were extracted withethyl acetate (3×150 mL). The combined extracts were washed with 1Mcitric acid (3×100 mL), water, 2M KHCO₃ (3×100 mL), water, and brine.The resulting solution was dried over sodium sulfate, filtered, andevaporated to give 29.1 g (72%) of the blocked tetrapeptide as a whitesolid (R_(f) =0.97, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tetrapeptide (29.1 g,28.0 mmol) in 35% trifluoroacetic acid/dichloromethane (300 mL) wasstirred 1/2 h at room temperature. The resulting solution wasconcentrated in vacuo and neutralized with 2M aqueous potassiumbicarbonate. The product was extracted into ethyl acetate (3×150 mL).The combined extracts were dried over sodium sulfate, filtered, andevaporated to give 28.4 g (ca. 100%+entrained solvent) of the free-aminocompound as a white solid.

To a stirred solution of N-CBZ-glycine (7.32 g, 35.0 mmol) andN-methylmorpholine (4.2 mL, 3.9 g, 38.1 mmol) in tetrahydrofuran (100mL) at -15° C. was added isobutyl chloroformate (4.8 mL, 5.0 g, 36.7mmol). After 2 min. a solution of the suitably protected tetrapeptide(26.3 g, 28.0 mmol) in 1:1 tetrahydrofuran/dimethylformamide (50 mL) wasadded. The reaction mixture was stirred at -15° C. for 1.5 h and thenallowed to warm to 0° C. At this time the reaction was quenched by theaddition of 2M aqueous potassium bicarbonate. The products wereextracted with ethyl acetate (3×150 mL). The combined extracts werewashed with 1M citric acid (3×100 mL), water, 2M KHCO₃ (3×100 mL),water, and brine. The resulting solution was dried over sodium sulfate,filtered, and evaporated to give 27.3 g (87%) of the blockedpentapeptide as a white solid (R_(f) =0.95, 10%methanol/dichloromethane).

To a suspension of the blocked pentapeptide (27.3 g, 24.2 mmol) inethanol (200 mL) was added water, until the mixture became very turbid(about 100 mL). The resulting mixture was shaken with palladium chloride(4.3 g, 24.4 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. Thereaction mixture became clear within about 1/2 h, which may indicatecompletion of the reaction. The catalyst was removed by filtration andthe filtrate was evaporated to give 14.6 g (82%) of the desiredpentapeptide dihydrochloride as a free-flowing white powder: [a]_(D)-12.1° (c 2.0, methanol).

EXAMPLE 8 Synthesis of Glycyl-L-Arginyl-L-Lysine

To a stirred solution of N^(a) -BOC-N^(g) -nitro-L-arginine (8.0 g, 25.0mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.3 mmol). After 2 min. a solution of L-(N^(im) -CBZ)lysinebenzyl ester hydrochloride (10.2 g, 25.0 mmol) and N-methylmorpholine(2.8 mL, 2.5 g, 25.0 mmol) in tetrahydrofuran (30 mL) was added. Thereaction mixture was stirred at -15° C. for 1.5 h and then allowed towarm to 0° C. At this time the reaction was quenched by the addition of2M aqueous potassium bicarbonate. The products were extracted with ethylacetate (3×15 mL). The combined extracts were washed with 1M citric acid(3×100 mL), water, 2M KHCO₃ (3×100 mL), water, and brine. The resultingsolution was dried over sodium sulfate, filtered, and evaporated to give16.3 g (97%) of the blocked dipeptide as a white solid (R_(f) =0.57, 10%methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected dipeptide (16.3 g, 24.3mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirredfor 1/2 h at room temperature. The resulting solution was concentratedin vacuo and neutralized with 2M aqueous potassium bicarbonate. Theproduct was extracted into ethyl acetate (3×100 mL). The combinedextracts were dried over sodium sulfate, filtered, and evaporated togive 17.0 g (ca. 100%+entrained solvent) of the free-amino compound as awhite semi-solid: R_(f) =0.12 (10% methanol/dichloromethane).

To a stirred solution of CBZ-glycine (7.32 g, 35.0 mmol) andN-methylmorpholine (4.2 mL, 4.0 g, 38.5 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (4.8 mL, 5.0 g, 36.8 mmol).After 2 min. a solution of the protected dipeptide (13.9 g, 24.3 mmol)in tetrahydrofuran (50 mL) was added. The reaction mixture was stirredat -15° C. for 1.5 h and then allowed to warm to 0° C. At this time thereaction was quenched by the addition of 2M aqueous potassiumbicarbonate. The products were extracted with ethyl acetate (3×150 mL).The combined extracts were washed with 1M citric acid (3×100 mL), water,2M KHCO₃ (3×100 mL), water, and brine. The resulting solution was driedover sodium sulfate, filtered, and evaporated to give 17.7 g (95%) ofthe blocked tripeptide as a white solid (R_(f) =0.51, 10%methanol/dichloromethane).

To a suspension of the blocked tripeptide (17.7 g, 23.2 mmol) in ethanol(250 mL) was added water, until the mixture became very turbid (about100 mL). The resulting mixture was shaken with palladium chloride (4.25g, 24.0 mmol) under an atmosphere of hydrogen (5 atm) for 18 h. Thecatalyst was removed by filtration and the filtrate was evaporated togive a white semi-solid. This material was dissolved in water, filteredthrough 0.45 m nylon syringe filters, and lyophilized to give 10.2 g(ca. 100%) of the desired tripeptide dihydrochloride as a white powder:[a]_(D) -14.6° (c 2, water); ¹ H NMR (500 MHz, D₂ O) d 8.81(1H, br s),8.30(1H, br s), 7.92(1H, br s), 4.37(1H, br s), 3.96(1H, d, J=4.8),3.58(2H, d, J=8.8), 3.13(2H, br s), 2.74(2H, br s), 1.90-1.20(10H, m);¹³ C NMR (125 MHz, D₂ O) d 175.2, 170.5, 166.9, 157.5, 115.0, 53.7,52.6, 31.4, 29.2, 27.8, 26.8, 25.0, 22.5, 19.1.

EXAMPLE 9 L-Alanyl-L-Histidyl-L-Lysine

AHK may be obtained as an acetate salt from Bachem Bioscience Inc.,Philadephia, Pa. (Catalog No. #-1555). Alternatively, AHK may besynthesized as the dihydrochloride salt by the following procedure.

To a stirred solution of N^(a) -BOC-N^(im) -CBZ-L-histidine (9.74 g,25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) intetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4mL, 3.6 g, 26.3 mmol). After 2 min. (N-ε-CBZ)-L-lysine benzyl esterhydrochloride (10.2 g, 25.0 mmol) was added. The reaction mixture wasstirred at -15° C. for 1.5 h and then allowed to warm to 0° C. At thistime the reaction was quenched by the addition of 2M aqueous potassiumbicarbonate. The products were extracted with ethyl acetate (3×150 mL).The combined extracts were washed with 1M citric acid (3×100 mL), water,2M KHCO₃ (3×100 mL), water, and brine. The resulting solution was driedover sodium sulfate, filtered, and evaporated to give 17.2 g (93%) ofthe blocked dipeptide as a white semi-solid (R_(f) =0.61, 10%methanol/dichloromethane), which was used in the followingtransformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (17.2 g, 23.2mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred1/2 h at room temperature. The resulting solution was concentrated invacuo and neutralized with 2M aqueous potassium bicarbonate. The productwas extracted into ethyl acetate (3×150 mL). The combined extracts weredried over sodium sulfate, filtered, and evaporated to give 16.8 g (ca.100%+entrained solvent) of the free-amino compound as a white solid:R_(f) =0.26 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-L-alanine (6.28 g, 25.0 mmol) andN-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL)at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol).After 2 min. a solution of the above protected dipeptide (14.9 g, 23.2mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture wasstirred at -15° C. for 1.5 h and then allowed to warm to 0° C. At thistime the reaction was quenched by the addition of 2M aqueous potassiumbicarbonate. The products were extracted with ethyl acetate (3×150 mL).The combined extracts were washed with 1M citric acid (3×100 mL), water,2M KHCO₃ (3×100 mL), water, and brine. The resulting solution was driedover sodium sulfate, filtered, and evaporated to a syrup, from which theblocked tripeptide was precipitated by dilution with 95% ethanol (300mL). The resulting material was collected on a filter, washed with 95%ethanol and dried to give a white solid: (R_(f) =0.49, 10%methanol/dichloromethane); mp 151-153° C.

To a suspension of the blocked tripeptide (21.5 g, 21.9 mmol) in ethanol(200 mL) was added water (about 200 mL). The resulting mixture wasshaken with palladium chloride (4.25 g, 24.0 mmol) under an atmosphereof hydrogen (5 atm) for 1 h. The resulting mixture, in which the bulk ofthe material (other than the catalyst) became dissolved, was filteredand the filtrate was concentrated in vacuo to remove volatile organics.The remaining aqueous solution was lyophilized to give 10.88 g of awhite solid. This material was dissolved in water, filtered through a0.2 m nylon membrane, and, again, lyophilized to give 10.50 g (99%) ofthe desired tripeptide dihydrochloride as a white powder: [a]_(D) -4.43°(c 3, H₂ O); ¹ H NMR (500 MHz, DMSO-d₆) d 8.73 (1H, d, J=7.8), 8.45 (1H,d, J=7.5), 8.09 (1H, s), 7.08 (1H, s), 4.59 (1H, dd, J=5.4, 7.5), 4.12(1H, m), 3.88 (1H, q, J=6.9), 3.03 (1H, dd, J=15.0, 4.8), 2.96 (1H, dd,J=15.0, 7.7), 2.74 (2H, t, J=7.5), 1.76-1.68 (1H, m), 1.66-1.51 (3H, m),1.41-1.21 (2H, m), 1.32 (3H, d, J=7.0); ¹³ C NMR (125 MHZ, DMSO-d₆) d174.0, 169.9, 169.5, 134.2, 130.5, 117.8, 52.6, 52.5, 48.0, 38.4, 30.3,28.2, 26.5, 22.4, 17.2.

EXAMPLE 10 Synthesis of Peptide-Copper Complexes at Various Molar Ratiosof Peptide to Copper

A. Peptide-Copper Complex at a 2:1 Molar Ratio

A solution of AHK was prepared by dissolving 2.6954 (0.0065 mole) of theAHK acetate (Bachem Bioscience Inc.) in approximately 10 ml of distilledwater. The initial pH of this AHK solution was 6.71. Separately, asolution of copper(II) chloride was prepared by dissolving 0.4479 gm(0.0033 mole) of anhydrous copper(II) chloride in approximately 2.0 mlof distilled water. The copper(II) chloride solution was slowly added tothe rapidly stirring AHK solution and the pH was constantly monitoredwith a pH meter. After all the copper(II) chloride solution was added,the combined solution pH was 3.83. The pH was then adjusted to 7.16 bythe slow addition of a solution of 0.5 M NaOH and the final volume wasadjusted to 20.0 ml by addition of distilled water. This procedureyielded an aqueous solution containing AHK:Cu at a molar ratio ofpeptide to copper of 2:1, and at a concentration of 10 mg/ml. Thesolution was a dark blue-purple and had a characteristic absorptionmaximum at 563 to 580 mn.

B. Peptide-Copper Complex at a 2:1 Molar Ratio

AHK was prepared as the dihydrochloride salt as described in Example 9.A solution of AHK was prepared by dissolving 0.6388 gm (0.00146 mole) ofL-alanyl-L-histidyl-L-lysine hydrochloride in approximately 5 ml ofdistilled water. The initial pH of this AHK solution was 2.45.Separately, a solution of copper(II) chloride was prepared by dissolving0.0967 gm (0.0007 mole) of anhydrous copper(II) chloride inapproximately 1.0 ml of distilled water. The copper(II) chloridesolution was slowly added to the rapidly stirring AHK solution and thepH was constantly monitored with a pH meter. After all the copper(II)chloride solution was added, the combined solution pH was 2.36. The pHwas then adjusted to 7.05 by the slow addition of a solution of 0.5 MNaOH, and the final volume was adjusted to 20.0 ml by addition ofdistilled water. This procedure yielded an aqueous solution containingAHK:Cu at a molar ratio of peptide to copper of 2:1, and at aconcentration of 10 mg/ml. The solution was a dark blue-purple and had acharacteristic absorption maximum at 563 to 580 nm.

C. Peptide-Copper Complex at a 1.1:1 Molar Ratio

AHK was prepared as the dihydrochloride salt as described in Example 9.A solution of AHK was prepared by dissolving 1.6144 gm (0.0037 mole) ofL-alanyl-L-histidyl-L-lysine hydrochloride in approximately 10 ml ofdistilled water. The initial pH of this AHK solution was 2.70.Separately, a solution of copper(II) chloride was prepared by dissolving0.4267 gm (0.0032 mole) of anhydrous copper(II) chloride inapproximately 2.0 ml of distilled water. The copper(II) chloridesolution was slowly added to the rapidly stirring AHK solution and thepH was constantly monitored with a pH meter. After all the copper(II)chloride solution was added, the combined solution pH was 2.14. The pHwas then adjusted to 6.89 by the slow addition of a solution of 0.5 MNaOH, and the final volume was adjusted to 20.0 ml by addition ofdistilled water. This procedure yielded an aqueous solution containingAHK:Cu at a molar ratio of peptide to copper of 1.1:1, and at aconcentration of 7.5 mg/ml. The solution was a dark blue-purple and hada characteristic absorption maximum at 593 nm, and a broad peak at 586to 607 nm.

D. Peptide-Copper Complex at a 1:1 Molar Ratio

A solution of AHK was prepared by dissolving 1.3007 gm (0.0007 mole) ofAHK acetate (Bachem Biosceince Inc.) in approximately 5 ml of distilledwater. The initial pH of this AHK solution was 6.95. Separately, asolution of copper(II) chloride was prepared by dissolving 0.0966 gm(0.0007 mole) of anhydrous copper(II) chloride in approximately 2.0 mlof distilled water. The copper(II) chloride solution was slowly added tothe rapidly stirring AHK solution and the pH was constantly monitoredwith a pH meter. After all the copper(II) chloride solution was added,the combined solution pH was 2.91. The pH was then adjusted to 7.08 bythe slow addition of a solution of 0.5 M NaOH, and the final volume wasadjusted to 15.0 ml by addition of distilled water. This procedureyielded an aqueous solution containing AHK:Cu at a molar ratio ofpeptide to copper of 1:1, and at a concentration of 10 mg/ml. Thesolution was a dark blue-purple and had a characteristic absorptionmaximum at 595 nm, and a broad peak at 584 to 612 nm.

EXAMPLE 11 Stimulation of Hair Growth by Representative Copper-PeptideComplexes

The following example illustrates the stimulation of hair growth inwarm-blooded animals after intradermal injection of representativepeptide-copper complexes of this invention.

In this experiment, the backs of C3H mice (60 days old, telogen hairgrowth phase) were closely clipped on day 1 using an electric clipper. Asterile saline solution containing the indicated peptide-copper complexwas then injected intradermally (i.e., infiltrated under the skin) attwo locations within the clipped areas of the mice. Injection at twolocations provided two test locations within the clipped area of eachmouse. Each injection (0.1 ml) contained between 0.36 to 0.55 mg of thepeptide-copper complex within the sterile saline solution. A group ofsaline injected mice (0.1 ml) served as controls. Following injection ofthe peptide-copper complexes, indications of hair growth were seenwithin 10 days. The first visual signs were a darkening of the skin in acircular region surrounding the injection site. The size of this regionis generally dose dependent, increasing with an increase in dose. The0.1 ml injections used in this experiment produced a circle of hairgrowth measuring approximately 0.5 cm² to 5.0 cm² in diameter. Activehair growth occurred between 14-20 days following injection, with amaximum effect seen by day 29. Both the number of mice growing hair atthe injection site and the diameter of the hair growth region weredetermined at day 21. A positive response was expressed as the number ofmice exhibiting hair growth at the injection sites compared to the totalnumber of mice injected in the study. The results of this experiment arepresented in Table 4 below (the day of onset is the day at which hairfollicle pigmentation was first observed):

                  TABLE 4                                                         ______________________________________                                        Stimulation of Hair Growth by Peptide-Copper Complexes                                     Molar             Number                                                      Ratio    Dose     of Animals                                     Peptide-Copper                                                                             (peptide (mg/     Growing Day of                                 Complex      to copper)                                                                             injection)                                                                             Hair    Onset                                  ______________________________________                                        GHKF:Cu      2:1      0.36 mg  4/5     10                                     PHKF:Cu      2:1      0.43 mg  5/5     10                                     (N-methyl)GHKVFV:Cu                                                                        2:1      0.55 mg  5/5     10                                     GHKVF:Cu     2:1      0.43 mg  5/5     10                                     SALINE       --       --       0/5     NA                                     ______________________________________                                    

EXAMPLE 12 Stimulation of Hair Growth by Representative Peptide-CopperComplexes

The following example illustrates the stimulation of hair growth inwarm-blooded animals after intradermal injection of representativepeptide-copper complexes of this invention.

As in Example 11 above, the backs of C3H mice (60 days old, telogen hairgrowth phase) were closely clipped on day 1 using an electric clipper. Asterile saline solution containing the indicated peptide-copper complexwas then injected intradermally (i.e., infiltrated under the skin) attwo locations within the clipped areas of the mice. Injection at twolocations provided two test locations within the clipped area of eachmouse. Each injection (0.1 ml) contained between 0.75 to 1.5 mg of thepeptide-copper complex within the sterile saline solution. A group ofsaline injected mice (0.1 ml) served as controls. Following injection ofthe peptide-copper complexes, indications of hair growth were seenwithin 10 days. The first visual signs were a darkening of the skin in acircular region surrounding the injection site. The size of this regionis generally dose dependent, increasing with an increase in dose. The0.1 ml injections used in this experiment produced a circle of hairgrowth measuring approximately 0.5 cm² to 5 cm² in diameter. Active hairgrowth occurred between 14-20 days following injection, with a maximumeffect seen by day 29. Both the number of mice growing hair at theinjection site and the diameter of the hair growth region weredetermined at day 21. A positive response was expressed as the number ofmice exhibiting hair growth at the injection sites compared to the totalnumber of mice injected in the study. The results of this experiment arepresented in Table 5.

                  TABLE 5                                                         ______________________________________                                        Stimulation of Hair Growth by Peptide-Copper Complexes                        Peptide-                                                                             Molar Ratio                                                                             Dose      Number of                                                                              Area of                                   Copper (peptide to                                                                             (mg/      Animals  Hair                                      Complex                                                                              copper)   injection)                                                                              Growing Hair                                                                           Growth                                    ______________________________________                                        PHK:Cu 2:1       1.00      2/5      >1 cm diameter                            GHL:Cu 2:1       1.50      3/4      >1 cm diameter                            GHE:Cu 2:1       1.50      2/4      >1 cm diameter                            PHA:Cu 2:1       1.50      1/4      <1 cm diameter                            PHF:Cu 2:1       0.75      4/4      >1 cm diameter                            PHL:Cu 2:1       1.50      2/4      <1 cm diameter                            AHK:Cu 2:1       0.75      1/4      <1 cm diameter                            AHK:Cu 2:1       1.50      4/4      >1 cm diameter                            VHK:CU 2:1       0.75      3/4      <1 cm diameter                            VHK:CU 2:1       1.50      4/4      >1 cm diameter                            ______________________________________                                    

EXAMPLE 13 Stimulation of Hair Growth by Peptide-Copper ComplexesContaining D-Amino Acids

This example illustrates the stimulation of hair growth in warm-bloodedanimals by intradermal injection of AHK:Cu (1.1:1) utilizing a D-aminoacids inplace of the naturally occuring L-amino acid.

In this experiment, the backs of C3H mice (60 days old, telogen hairgrowth phase) were closely clipped on day 1 using an electric clipper. Asterile saline solution containing AHK:Cu (1.1:1), or AHK:Cu (1.1:1)containing a D-amino acid, was then injected intradermally (i.e.,infiltrated under the skin) at two locations within the clipped areas ofthe mice. Injection at two locations provided two test locations withinthe clipped area of each mouse. Each injection (0.1 ml) contained either1.2 or 1.8 μmoles per injection of peptide-copper complex in the sterilesaline solution. A group of saline injected mice (0.1 ml) served ascontrols. Following injection of peptide copper complex, indications ofhair growth were seen within 10 days. The first visual signs were adarkening of the skin in a circular region surrounding the injectionsite. The size of this region is generally dose dependent, increasingwith an increase in dose. The 0.1 ml injections used in this experimentproduced a circle of hair growth measuring approximately 0.5 cm² to 5cm² in diameter. Active hair growth occurred between 14-20 daysfollowing injection, with a maximum effect seen by day 29.

The degree of hair growth was determined by measuring the total area ofhair growth at the two injection sites. The data from this experiment ispresented in Table 6.

                  TABLE 6                                                         ______________________________________                                        Stimulation of Hair Growth by Peptide-Copper Complexes                        Containing D-Amino Acids                                                               Molar Ratio                                                                              Dose                                                      Peptide-Copper                                                                         (peptide to                                                                              (μmoles per                                            Complex  copper)    injection)                                                                              Area of Hair Growth                             ______________________________________                                        AHK:Cu   1.1:1      1.2       3.07 ± 0.76                                  AHK:Cu   1.1:1      1.8       3.24 ± 1.17                                  AH-(D)K:Cu                                                                             1.1:1      1.2       3.30 ± 0.30                                  AH-(D)K:Cu                                                                             1.1:1      1.8       3.94 ± 0.35                                  (D)A-HK:Cu                                                                             1.1:1      1.2       1.88 ± 0.57                                  (D)A-HK:Cu                                                                             1.1:1      1.8       2.68 ± 0.49                                  ______________________________________                                    

The table above illustrates that the substiturion of D-amino acids for acorresponding L-amino acids dose not effect the hair growth activity ofthe peptide copper complexes.

EXAMPLE 14 Stimulation of Hair Growth by Topical Application of aPeptide-copper Complex

This example illustrates the stimulation of hair growth in warm-bloodedanimals by topical application of a peptide-copper complex In thisexperiment, telogen cycle female C3H mice (60-65 days old) were preparedby clipping their posterior dorsal region (i.e., day 1). Topicalapplication of peptide-copper complexes was performed twice per day(Monday-Friday) using a cotton-tipped applicator which deliveredapproximately 0.1 ml per treatment. The topical formulation used in thisexperiment contained the following components:

    ______________________________________                                        Peptide copper Complex                                                                            0.1-0.5%  (w/w)                                           Sterile Water       16.9-16.5%                                                                              (w/w)                                           Propylene Glycol    50.0%     (w/w)                                           Ethanol             30.0%     (w/w)                                           Nonoxynol-9         3.0%      (w/w)                                           ______________________________________                                    

Topical application of the above formulation continued until the onsetof follicle pigmentation, which proceeds the emergence of the hairshaft. Measurement of the degree of response was performed using digitalimage analysis at weekly intervals, beginning at day 14. Data wasexpressed as the percent treatment area response using the followingequation:

    % treatment area=(growth area/treatment area)×100

For comparison purposes to illustrate the effect of hydrophobic aminoacid residues on hair growth after topical application, AHK:Cu wascompared to AHF:Cu. In this experiment, topical formulations containingAHK:Cu (1.1:1) and AHF:Cu (1.1:1) were prepared at a concentration of0.5% and 0.1% (w/w) as indicated above. Hair growth response (i.e.,"Percent Treatment Area") was determined at day 20, day 27 and at day34. The results of this experiment are presented in Table 7.

                  TABLE 7                                                         ______________________________________                                        Peptide-                                                                              Molar Ratio                                                           Copper  Peptide to                                                                              Concentration    Percent Treatment                          Complex copper)   (% w/w)     Day  Area                                       ______________________________________                                        AHK:Cu  1.1:1     0.1%        20   1.29 ± 1.29                             AHK:Cu  1.1:1     0.1%        27   23.07 ± 18.84                           AHK:Cu  1.1:1     0.1%        34   90.14 ± 2.96                            AHK:Cu  1.1:1     0.5%        20   75.87 ± 7.64                            AHK:Cu  1.1:1     0.5%        27   100                                        AHK:Cu  1.1:1     0.5%        34   100                                        AHF:Cu  1.1:1     0.1%        20   0.00                                       AHF:Cu  1.1:1     0.1%        27   0.00                                       AHF:Cu  1.1:1     0.1%        34   12.91 ± 12.91                           AHF:Cu  1.1:1     0.5%        20   55.05 ± 17.44                           AHF:Cu  1.1:1     0.5%        27   100                                        AHF:Cu  1.1:1     0.5%        34   100                                        ______________________________________                                    

The data presented in Table 7 illustrates that peptide-copper complexescontaining hydrophilic residues (i.e., lysine amino acid of AHK:Cu) aremore active in stimulating hair growth than similar peptides containinghydrophobic amino acid residues (i.e., the phenylalanine amino acid ofAHF:Cu) following administration by topical administration. This is incontrast to administration by injection where peptide-copper complexescontaining hydrophilic residues are less active than than similarpeptides containing hydrophobic amino acid residues.

EXAMPLE 15 Stimulation of Hair Growth by Intraperitoneal Injection ofPeptide-Copper Complexes

The following experiment illustrates the maintenance of hair follicleviability (i.e., growth) by intraperitoneal (systemic) injection of thepeptide-copper complex GHKVFV:Cu during treatment with thechemotherapeutic agent cytosine arabinoside (Ara-C).

In this experiment, Sprague-Dawley rat pups (age 8 days) were maintainedin 4 litters (n=10/litter) for the duration of this study. On day 0,litters received intraperitoneal (IP) injections of GHKVFV:Cu (2:1) in asterile saline solution, or a saline control (1 injection per animal,0.1 ml per injection). On day 1, all animals began a series of 7consecutive daily IP injections with Ara--C (50 mg/kg). On day 8, allanimals were evaluated for the extent of hairloss (alopecia) using thefollowing rating scale:

    ______________________________________                                        Grade            Degree of Alopecia                                           ______________________________________                                        0                Normal (no loss of hair)                                     1                Slight thinning                                              2                Moderate thinning                                            3                Sparse hair cover                                            4                Total loss of hair                                           ______________________________________                                    

Ara-C injections caused significant hair loss by day 5-6 in mostanimals. In order to evaluate the effect of GHKVFV:Cu, the degree ofhairloss was evaluated daily. Injection of GHKVFV:Cu at a dosage of 50mg/kg caused a mild retention of hair on the body of the test animals.This was primarily seen on the head, with sparse remaining hair on thebody. This was in contrast to the saline control (+Ara--C) group whichshowed total hair loss. Table 8 presents the results of this experimentas evaluated on day 8 using the previously described rating scale, withthe "Degree of Alopecia" being expressed as the average response for allanimals.

                  TABLE 8                                                         ______________________________________                                                     Dose per Animal                                                  Peptide-Copper                                                                             injection                                                                              Dosage       Degree of                                  Complex      (mg)     (mg/kg)  n = Alopecia (mean)                            ______________________________________                                        Saline Only  --       0.0      10  0.0                                        Saline + Ara-C                                                                             --       0.0      10  4.0                                        GHKVFV:Cu + Ara-C                                                                          100      50       10  3.0                                        ______________________________________                                    

The observation of retained hair was confirmed histologically on day 8.Of the animals recieving 50 mg/kg of GHKVFV:Cu, approximately 30-40% ofdorsal hair was found to be in anagen, compared to 5-10% for animalsreceiving saline+Ara--C alone. Saline control animals not recievingAra--C had 100% anagen follicles.

Example 16 Stimulation of Hair Growth by Intradermal Injection ofPeptide-Copper Complexes

The following experiment illustrates the localized maintenance of hairfollicle viability (i.e., growth) by intradermal (local) injection ofthe peptide-copper complex AHK:Cu during treatment with thechemotherapeutic agent cytosine arabinoside (Ara--C).

In this experiment, Sprague-Dawley rat pups (age 8 days) were maintainedin 5 litters (n=10-11/litter) for the duration of this study. On day 0,litters received intradermal (ID) injections of AHK:Cu (1:1) in asterile saline solution, or a saline control (1 injection per animal,0.05 ml per injection). Each liter contained 2 normal control animalswhere no AHK:Cu or Ara--C was administered (i.e., saline only). On day1, designated animals began a series of 7 consecutive dailyintraperitoneal (IP) injections with Ara--C (25 mg/kg). On day 10, allanimals were evaluated for the extent of hairloss (alopecia) at theinjection sites using the rating identified in Example 15.

Ara--C injections caused significant hair loss by day 5-6 in mostanimals. In order to evaluate the stimulatory effect of AHK:Cu, thedegree of hairloss was evaluated at the injection site daily. AHK:Cuinjection generally caused a retention of hair in a 0.25 cm radiusaround the injection site, most notably in the 0.1 to 0.5 mg dosegroups. Table 9 presents the results as evaluated on day 10 using thepreviously described rating scale, with the the "Degree of Alopecia"being expressed as the average response seen at the site of injection.

                  TABLE 9                                                         ______________________________________                                                              Animal                                                  Peptide-Copper                                                                           Dose per   Dosage       Degree of                                  Complex    injection (mg)                                                                           (mg/kg)  n = Alopecia (mean)                            ______________________________________                                        Saline Only                                                                              --         0.0      8   0.00                                       Saline + Ara-C                                                                           --         0.0      8   4.00                                       AHK:Cu + Ara-C                                                                           0.05       35       8   3.25                                       AHK:Cu + Ara-C                                                                           0.10       7.0      8   2.38                                       AHK:Cu + Ara-C                                                                           0.25       17.5     9   1.44                                       AHK:Cu + Ara-C                                                                           0.50       35.0     9   1.11                                       ______________________________________                                    

The observation of retained hair within the area of AHK:Cu injection wasexamined histologically. While normal appearing and functioning anagenhair follicles were seen at the injection site of AHK:Cu, follicleslocated away from the injection were dystrophic and non-functional(disruption of the integrity of inner and outer root sheaths, anddisplaced hair shafts). These data confirm the gross observations ofnormal hair follicle function within the site of AHK:Cu injection, andillustrate the stimulatory effect of AHK-Cu on the hair follicle whichmaintains the active growth cycle during chemotherapy treatment.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notto be limited except as by the appended claims.

We claim:
 1. A method for stimulating hair-growth on an animal in needthereof, comprising administering to the animal an effective amount of apeptide-copper complex having the structure:

    [R.sub.1 --R.sub.2 --R.sub.3 ]:copper                      (II)

wherein R₁ is an amino acid or amino acid derivative, R₂ is histidine orarginine; and R₃ is a chemical moiety joined to R₂ by an amide bond,wherein R₃ is --NH₂, an alkylamino moiety having from 1-20 carbon atomsor an arylamino moiety having from 6-20 carbon atoms.
 2. The method ofclaim 1 wherein R₁ is an amino acid.
 3. The method of claim 1 wherein R₂is histidine.
 4. The method of claim 1 wherein R₂ is arginine.